Tubocurarine antigens and antibodies specific therefor

ABSTRACT

Antigens are prepared by coupling tubocurarine derivatives to immunogenic carrier materials. The coupling is effected via a linking group derived from an amino acid involving an azo link to the tubocurare moiety and an amide linkage to the immunogenic carrier material. In preferred embodiments, proteins are used as the carrier materials. The resulting antigens produce immunological effects when injected into host animals, including the formation of antibodies specific for tubocurarine components in biological fluids.

United States Patent 1191 Spector 1451 May 7,1974 1 [75] Inventor:

[73] Assignee: Hoffmann-La Roche Inc., Nutley,

22 Filed: Mar. 10, 1972 21 Appl. No.: 233,814

Sidney Spector, Livingston, NJ.

[56] References Cited UNITED STATES PATENTS l H1945 Friedheim 260/112 X10/1960 Campbell 424/12 OTHER PUBLICATIONS Chem. Abstracts, Vol. 52,1958, l4845c-d, Aladjemo et al.

Chem. Abstracts, Vol. 57, 1962, '4833e-f, Ehren preis PrimaryExaminer-Howard E. Schain Attorney, Agent, or Firm-Samuel L. Welt; JonS.

Saxe; Alan M. Krubiner [5 7] ABSTRACT Antigens are prepared by couplingtubocurarine derivatives to immunogenic carrier materials. The couplingis effected via a linking group derived from an amino acid involving anazo link to the tubocurare moiety and an amide linkage to theimmunogenic carrier material. In preferred embodiments, proteins areused as the carrier materials. The resulting antigens produceimmunological effects when injected into host animals, including theformation of antibodies specific for tubocurarine components inbiological fluids.

6 Claims, No Drawings 1 TUBOCURARINE ANTIGENS AND ANTIBOD SPECIFICTHEREFOR BACKGROUND OF THE INVENTION levels of tubocurare derivativespresent in a given subject are not very sensitive and are at best rathercrude and subjective. A more highly sensitive assay for the presence oftubocurarine derivatives in a biological system would represent anextremely important advance in the art. I

It has been known in the art for some time that various small molecules(haptens), which by themselves are wholly devoid of antigenicity, canmodify the antigenic properties of a protein when a small molecule iscombined with the protein through stable covalent linkages. In U.S. Pat.No. 2,372,066, patented Mar. 20, 1945, it is disclosed that antigens maybe prepared by combining histamine or histamine-like compounds bylinking the imidazole ring to a desired protein through aradicalcontaining a group capable of coupling with the protein. Theseantigens are used either by direct injection into a subject wherebyresistance, refractoriness or active immunity is developed in saidsubject or for injecting into host animals from which antibodiesspecific to the hapten moiety, e.g., the histamine or histamine-likesubstance are developed.

A similar contemporarydisclosure was made by Lansteiner in theSpecificity of Serological Reactions," Harvard University Press,Cambridge, Mass. 1945) wherein p-amino benzene arsonic acid was coupledto a protein via its" diazonium saltto form a chemically simple,well-defined compound which was antigenic and stimulated the productionof antibodies. Furthermore, the antibodies to this immunogen (conjugatedprotein) can combine with the small molecule, e.g., the arsonic acidwhich is unattached to any protein. This antibody is quite specific inactivity. For example, if an isomer of arsonilic acid, in which the AsOH group is in the meta position relative to the amino group, isutilized, it will not combine with they antibody formed against theprotein-arsonilic acid complex in which the AsO H group is para to theamino group.

It should be mentioned that it is not yet possible, in the present stateof the art, to predict ordetermine what properties are required toenable a molecule to act as an antigen. At one time, molecular weightand the possession of an aromatic group were thought to be the decidingfactors. With time, the critical molecular weight required forantigenicity has been remarkably reduced. lt is still believed, however,that the molecular weight will, to some extent, determine the antigeniccapabilities of a molecule. Other factors such as molecular shape andchemical reactivity must also play a role in the antigenic propertiesand thus render prediction of such properties exceedingly moredifficult.

DETAILED DESCRIPTION OF THE INVENTION to an immunogenic carriermaterial. The coupling is effected via a linking group of the formulawherein m is an integer of from-O to 5.

This linking group is derivedv from an aminobenzoic acid (m is 0) or anaminophenyl lower alkanoic acid (m l to 5)..In preferred embodiments,the tubocurarine hapten is bonded via the linking group derived frompaminobenzoic acid, to a protein; Additionally, the present inventionrelates to antibodies which would complex with some specificity to thetubocurarine hapten. These antibodies are produced by treating hostanimals with the aforesaid antigen. Such specific antibodies'are readilyisolated from sera obtained from host animals after treatment of thesehost animals with the antigen.

As used herein, the term tubocurarine or tubocurarine derivative ismeant to include the vdand l-enantiomeric form of tubocurarine and itsmono? or di-O- lower alkyl ethers, the acid addition salts thereof,(e.g., monoor di-hydrochlorides or hydrobromides and the like); thequaternary ammonium salts thereof (e.g., mono-or di-methochlorides,methobromides, methiodides, and the like) and mixtures of the above.Preferred tubocurarine derivatives are those of the d-enantiomericseries, most preferably d tubocurarine chloride.

As used herein, the term immunogenic carrier material is meant toinclude those-materials which have the properties of independentlyeliciting an immunogenic response in a host animal when injectingtherein and which can be coupled 'via a peptide linkage to a moleculehaving a free or activated carboxyl group. Suitable carrier materialsinclude, for example, proteins; and natural or synthetic polymericcompounds such as polypeptides, e.g., polylysine; and the like.Particularly preferred carrier materials for the practiceof the presentinventionare proteins.

The identity of the protein material utilized in the preparation of apreferred antigen of the present invention is not critical. Examples ofpreferred proteins useful in the practice of. the present inventioninclude mammalian serum proteins, such as for example, human gammaglobulin, human serum albumin, bovine serum albumin, rabbit serumalbumin and bovine gamma globulin. Other suitable protein products willbe suggested to oneskilled in the art. It is generally preferred thatproteins be utilized which are foreign to the animal hosts in which theresulting antigen will be employed.

Tubocurarine hapten is coupled to the immunogenic carrier material,e.g., a protein, via a linking group de rived from an amino acid of theformula wherein m is as above.- Y H v g The linkage is effected to thetubocurarine via an azo bond. This bond is formed by diazotization ofthe amino group of the amino acid and coupling onto the aromatic ring ofthe tubocurarine hapten.

Diazotization may be carried out by any method known per se foreffecting such a reaction. For example, the amino acid can be diazotizedby reacting with a metal nitrite, for example, sodium nitrite, in thepresence of a mineral acid, for example, hydrochloric acid. Thediazonium salt thus formed can be .then coupled to the aromaticsubstrate, the tubocurarine hapten, by contacting the diazonium saltwith the tubocurarine. The coupling reaction is preferably effected at abasic pH, for example, from about pH 8 to about pH 12. After thecompletion of the reaction excess amino acid can be removed by acidextraction.

The tubocurarine coupledvia an azo linkage to the amino acid moiety, asdescribed above, hereinafter referred to as azo-tubocurarine, can thenbe coupled to the immunogenic carrier material, e'.g., a protein, to

form the antigen of the present invention. Any techniques well known inprotein and peptide chemistry'in establishing peptide bonds may be used.Thus, for example, one such technique would involve dissolving theprotein and a dehydrating agent in a suitable inert solvent followed byadding a large molar excess of the azo-tubocurarine. The reaction may beconducted at in the range of from about 4 to about 65. Upon completionof the reaction, the excess azo-tubocurarine molecules and dehydrationreagent may be removed by dialysis. The dialysis may be monitored bychecking the dialysate for the presence of azo-tubocurarine ordehydrating agent or, alternatively, may be conducted for apre-determined period oftime, e.g., 3 days. Purified antigen isrecovered as a residue in the dialysis ba g.

The dehydrating 1 agent which may be used in the aforesaid reaction willbe selected from those commonly employed in peptide chemistry forinitiating the formation of peptide bond. A particularly suitable groupof dehydrating agents comprise the carbodiimides, most preferablydicyclohexyl carbodiimide or l-ethyl-3-( 3-dimethylamino propyl)-carbodiimide. The amount molar excess of the hapten over the proteinin the aforesaid reaction will, of course, depend upon the identity ofthe hapten utilized. and the protein selected for the reaction.Generally, a molar excess in the range of from about 100 to about 1,000,most preferably in the range of from about 500 to about 1,000 will beutilized. It is generally found that from about 2 to about 3tubocurarine groups are added to a molecule of pro- .tein depending ofcourse on the amount of molar excess of tubocurarine used in thecoupling reaction.

Another useful technique for the preparation of the antigens of thepresent invention is tofirst form an activated derivative of thecarboxyl group of the azotubocurarine moiety and then to react saidactivated derivative with the protein to form the desired antigen.Suitable activated derivatives include activated esters,

such as 'p-nitrophenyl esters; acyl imidazoles; and so forth. Activatedester derivatives are conveniently prepared from the free'acid byreacting said free acid with the desired alcohol in the presence of asuitable dehydrating agent, such as'a carbodiimide, under reactionconditions similar to those described above.- Acyl imidazoles may beprepared by reacting-the free carboxyl fied by dialysis.

The antigens of the present invention may also be prepared by firstcoupling the amino acid via an amide linkage to the immunogenic carriermaterial, for example, a protein, and then coupling this proteinderivative,

having anaminophenyl group, to the'tubocurarine by diazotization of theamino group and coupling as described above. It is generally preferredto first couple the amino acid linking group to'the tubocurarine via theazo-linkage, followed by coupling of this azotubocurarine to the proteinby a peptide linkage.

The antigens of the present invention maybe utilized to induce formationof antibodies specific to tubocurarine derivatives, in the serum of hostanimals by injecting the antigen in such host repeatedly over a periodof time, collecting the serum, precipitating the antibody with a neutralsalt solution. and purifying the antibody by dialysis and columnchromatography. Suitable host animals for this purpose include mammals,such as rabbits, horses, goats, guinea pigs, rats, cows, sheep, etc. Theresulting antibodies will have a multiplicity of active sites which willselectively complex with tubocurarine derivatives and theabove antigens.

The formation of tubocurarine specific antibodies in host animals maybemonitored by taking blood samples from the host animals and adding to itan amount of the same tubocurarine-protein antigen used for inoculation.The presence of a precipitate indicates antibody activity. The antigentreatments of the animals can be continued until the antibody titerreaches the desired level of activity. For the purpose of thisapplication, the antibody titer is defined as being the maximumconcentration of protein precipitated after the addition ofvarying.knownconcentrations of antigen to fixed amounts of serum. I I

The tubocurarine specific antibodies can be isolated from the sera oftreated host animals by utilizing techniques well known in thebiochemical art. For example, the sera obtained from treated hostanimals can be acted upon by a neutral salt which will effectprecipitation of the desired tubocurarine specific. antibodies. Suitableneutral salts for this purpose include sodium sulfate, magnesiumsulfate, a sodium hydrogen phosphate mixture or ammonium sulfate. Theneutral salt preferred for the purpose of the present invention isammonium sulfate. Purification techniques subsequent to theprecipitation step may also be employed. For example, the obtainedantibodies may be further purified by subjecting such antibodies todialysis and column chromatography. The resulting antibody may becharacterized as being a gamma globulin fraction .protein having amolecular weight of about l60,000. This antibody will complex withtubocurarine and tubocurarine' antigens as described above.

Specific antibodies of the present invention are useful as reagents inbiochemical assays for the determination of the presence of tubocurarinederivatives in biological fluids. A particularly preferred assayprocedure is an immunoprecipitation procedure which can be used tomeasure nanogram amounts of tubocurarine derivatives in serum or urine.In such a procedure, a known amount of a labelledtubocurarine derivativeis mixed with the tubocurarine specific antibody and a sample containingthe unknown quantity of tubocurarine. The amount of tubocurarine in thesample can be determined by measuring the amount of competitiveinhibition observed between the binding of the labelled tubocurarine andthe sample with the specific antibody, and then calculating the amountof tubocurarine in the sample from a standard curve. Suitable labelledtubocurarine derivatives for this purpose include isotopically labelledtubocurarine, particularly those labelled with tritium and carbon-l4, aswell as tubocurarine labelled with an electron spin resonance group.Examples of the use of various electron spin resonance labelledmolecules in bioassays are to be found in US. Pat. Nos. 3,453,288,3,481,952, and 3,507,876.

The novel antigens and antibodies of the present invention may beutilized in conjunction with conventional additives, buffers,stabilizers, diluents, or in combination with other physiologicallyactive substances. The preparation and use of compositions in obtainingantigens and antibodies in conjunction with physiologically acceptableadjuvants are well known in the art.

This invention is further illustrated by the following specificexamples. All temperatures are expressed in degrees centigrade.

EXAMPLE 1 Preparation of antigen p-Aminobenzoic acid was dissolved inml. of 1N HCl at 4. A solution of 100 mg. sodium nitrite in 0.5 ml.water was added. The excess sodium nitrite was removed by the additionof 600 mg. ammonium sulfamate in 1 ml. water. The solution of diazotizedpaminobenzoic acid was added to a solution of 50 mg. d-tubocurarinechloride in 5 ml. 90 percent aqueous methanol. The pH was adjusted to 10and the diazonium coupling was allowed to proceed for 2 hours at 05. Themixture was acidified with 0.1N HCl to pH 1.5 to remove excessp-aminobenzoic acid; The precipitated azo-d-tubocurarine was dissolvedin 01 NaOH and the mixture was centrifuged to remove excessdtubocurarine. The azo-d-tubocurarine was extracted into a 4: 1 mixtureof benzene and methanol and the solvent was removed. The residue wasadded to a solution of 100 mg. dicyclohexyl carbodiimide dissolved in 10ml. 0.9 percent sodium chloride solution containing 100 mg. bovine serumalbumin. The mixture was allowed to stand overnight at 4 and was thendialyzed versus 0.9 percent sodium chloride for 3 days. The residueremaining in the dialysis bag was the azo-dtubocurarine-BSA-antigen.

EXAMPLE 2 Preparation of antibody Rabbits of the New Zealand albinostrain were immunized with 1 mg. of azo-d-tubocurarine-BSA-antigenprepared as in example 1. The immunogen was dissolved in phosphatebuffered saline, pH 7.4, emulsified with an equal volume of completeFreund adjuvant and injected into the footpads. Booster injections of100 pg. of antigen in adjuvant were weeks in the footpads and sides.

I Antiserum was collected 5 to 7 days after booster injections. Bloodcollected by cardiac puncture was incubated at 37 for 1 hour and thenkept overnight at 4. After centrifugation at 5,000 rpm for minutes 'at4, serum was separated from the clot.

EXAMPLE 3 Radioimmunoassay' The radioimmunoassay was performed byincubating 0.1 ml. of various dilutions of antiseraobtained in example 2in the presence of 0.1 ml. normal serum from the same animal species,0.18 ml. of 0.01N phosphate buffered saline, pH 7.2, and 0.025 ml. of asolution of d-tubocurarine dimethyl-C-ether iodide (Amersham- Searle, 1l2 uc/mg), showing 4,500 cpm, at 4 overnight. After incubation, aneutral saturated ammonium sulfate solution (volume equal to incubationmedium) was added to all tubes. The precipitate was sedimented bycentrifugation at 5,000 rpm for 15 minutes at 4 and was washed twice at50 percent ammonium given every 6 to 8 sulfate solution. The washedprecipitate, containing antibody-bound labelled d tubocurarine, wasdissolved in 0.5 ml. of commercial detergent solubilizer, such asNCS-solubilizer and quantitatively transferred and counted in a PackardTri-carb liquid scintillation spectrometer afterthe addition of 12 ml.of Brays scintillation solution. The tube which contained no unlabelledd-tubocurarine chloride served as a measure of maximum antibody-boundradioactivity. The addition of increasing amounts of unlabelledd-tubocurarine chloride to a fixed amount of labelled material andantiserum resulted in a competitive inhibition of the labelled materialfor the formation of the antibody-hapten complex. The data obtained issummarized below in Table 1.

TABLE 1 Nanograms non-radioactive d-tubocurarine added Percentinhibition binding of d-tubocurarine dimethyl C"-ether-iodidc 0.25 it)0.5 30 1.0 as 5.0 10.0

rine such as acetyl choline, succinyl choline, and neostigmine, were notbound by the antibody.

I claim:

1. An antigen consisting essentially of a tubocurarine derivativecoupled to the azo moiety of a linking group of the formula l0 Grimm-2L-N=N wherein m is an integer of from 0 to 5,

7- 8 7 said linking group being further coupled-by a peptide 4. Theantigen of claim 3 wherein said tubocurarine linkage to animmunogeniccarrier material sed i i i (Ltubocurarine hl i h lected from groupconsisting of P'Q and 5. The antigen of claim 1 wherein said immunogenicpolypeptides.

I carrier material is a protein. 2. Th ant of claim 1 wherein said lmkmrou is a group I g g p 6. The antigen of claim 5 wherein said protein ISbo- 3. The antigen of claim 1 wherein said tubocurarine V1116 Serumalbummderivative is of the d-enantiomeric series.

2. The antigen of claim 1 wherein said linking group is a p-azo-benzoylgroup.
 3. The antigen of claim 1 wherein said tubocurarine derivative isof the d-enantiomeric series.
 4. The antigen of claim 3 wherein saidtubocurarine derivative is d-tubocurarine chloride.
 5. The antigen ofclaim 1 wherein said immunogenic carrier material is a protein.
 6. Theantigen of claim 5 wherein said protein is bovine serum albumin.